Method of Preparing Piceatannol Using Bacterial Cytochrome P450 and Composition Therefor

ABSTRACT

Provided is a method of preparing piceatannol, and more particularly, to a method of preparing piceatannol from resveratrol using bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof, and a composition and a kit therefor.

TECHNICAL FIELD

The present invention relates to a method of preparing expensivepiceatannol from resveratrol using bacterial cytochrome P450 BM3(CYP102A1) or mutants thereof.

This work was supported in part by the 21C Frontier Microbial Genomicsand Application Center Program of the Ministry of Education, Science &Technology of the Republic of Korea [Project No.: MG08-0306-2-0, Title:Development of humanized bacterial monooxygenase for fine chemicalsusing microbial cytochrome P450 enzyme genomics].

BACKGROUND ART

Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin, which is anantitoxic substance produced by a plant tissue in response to externaltoxicity and found in a wide variety of dietary sources includinggrapes, plums, and peanuts. It exhibits beneficial effects includinganti-oxidant, anti-inflammatory, cardioprotective, and anti-tumoractivities (Kundu and Surh, 2008; Pirola and Fröjdö, 2008; Athar, etal., 2007). Currently, numerous preclinical findings suggest resveratrolas a promising nature's arsenal for cancer prevention and treatment. Asa potential anti-cancer agent, resveratrol has been shown to inhibit orretard the growth of various cancer cells in culture and implantedtumors in vivo. The biological activities of resveratrol are found torelate to its ability in modulating various targets and signalingpathway.

Piceatannol (3,5,3′,4′-tetrahydroxystilbene) is a polyphenol found ingrapes and other plants. It is known as a protein kinase inhibitor thatexerts immunosuppressive and antitumorigenic activities on several celllines, and has been shown to exert various pharmacological effects onimmune and cancer cells (Kim et al, 2008b and references therein). Inhumans, piceatannol is produced as a major metabolite of resveratrol byCYP1 B1 and CYP1A2 (Potter et al., 2002; Piver et al., 2004). Inaddition, the metabolism of trans-resveratrol into two majormetabolites, piceatannol (3,5,3′,4′-tetrahydroxystilbene) and anothertetrahydroxystilbene, was catalyzed by recombinant human CYP1A1, CYP1A2and CYP1 B1 (Piver et al., 2004).

Cytochrome P450 enzymes (P450s or CYPs) constitute a large family ofenzymes that are remarkably diverse oxygenation catalysts foundthroughout nature, from archaea to humans(http://drnelson.utmem.edu/CytochromeP450.html). Because of theircatalytic diversity and broad substrate range, P450s are attractive asbiocatalysts in the production of fine chemicals, includingpharmaceuticals (Guengerich 2002; Urlacher et al., 2006; Yun et al.,2007; Lamb et al., 2007). In spite of the potential use of mammalianP450s in various biotechnology fields, they are not suitable asbiocatalysts because of their low stability, catalytic activity, andavailability.

If a metabolite, such as piceatannol, has a biological activity, directadministration of the metabolite into a living body may be beneficial.However, large quantities of the metabolite need to be produced. Ifpro-drugs are converted to biologically ‘active metabolites’ by humanliver P450s during the drug development process (Johnson et al., 2004),large quantities of the pure metabolites are required to understand thedrug's efficacy, toxic effect, and pharmacokinetics.

The pure metabolites may be difficult to synthesize. An alternative tochemical synthesis is to use P450s to generate the metabolites of drugsor drug candidates. Hepatic microsomes can be a source of human P450s,but their limited availability make their use in preparative-scalemetabolite synthesis impractical. Some human enzymes can also beobtained by expression of recombinant hosts. Metabolite preparation hasbeen demonstrated using human P450s expressed in Escherichia coli and ininsect cells (Parikh et al., 1997; Rushmore et al., 2000; Vail et al.,2005), but these systems are costly and have low productivities due tolimited stabilities and slow reaction rates (usually <5 min⁻¹(Guengerich et al., 1996)). An alternative approach to preparing thehuman metabolites is to use an engineered bacterial P450 that has theappropriate specificity.

The P450 BM3 (CYP102A1) from Bacillus megaterium has strong similarityto eukaryotic members of the CYP4A (fatty acid hydroxylase) family. Itwas shown that engineered CYP102A1 mutants could oxidize several humanP450 substrates to produce the authentic metabolites with higheractivities (Kim et al., 2008; Otey et al., 2005; Yun et al., 2007 andreferences therein). Furthermore, CYP102A1 is a versatile monooxygenasewith a demonstrated ability to work on a diversity of substrates(Bernhardt et al., 2006, Di Nardo et al., 2007).

Recently, wild-type CYP102A1 has been engineered to oxidize compoundsshowing little or no structural similarity to its natural substratefatty acids (Lamb et al., 2007). The compounds include testosterone,several drug-like molecules, and polycyclic aromatic hydrocarbons(PAHs), which are known substrates of human P450 enzymes (Carmichael etal., 2001; van Vugt-Lussenburg et al., 2006). However, there has been noresearch on whether resveratrol can be used as a substrate. A set ofCYP102A1 mutants was shown to generate larger quantities of theauthentic human metabolites of drugs, which may be difficult tosynthesize (Otey et al., 2005). An alternative approach to preparing themetabolites is to use engineered CYP102A1 enzymes with desiredproperties.

Based on the scientific literature, several amino acid residues inCYP102A1 were mutated to generate mutant enzymes showing increasedactivity toward human P450 substrates (Yun et al., 2007). Very recently,it was reported that some selected mutations enabled the CYP102A1 enzymeto catalyze O-deethylation and 3-hydroxylation of 7-ethoxycoumarin,which are the same reactions catalyzed by human P450s (Kim et al.,2008a).

There are several patent applications related to piceatannol. That is, acomposition for antihypertensive effects comprising a Rhei Rhizomeextract or active compounds isolated therefrom is disclosed in KoreanPatent Application No. 10-2005-0126879, and a cosmetic compositioncontaining piceatannol and vitamin A is disclosed in Korean PatentApplication No. 10-2007-0025087. However, a patent application relatedto a method of preparing piceatannol has not been filed. While a methodof chemically synthesizing resveratrol and piceatannol is disclosed inWO2008012321, a method of biologically preparing resveratrol andpiceatannol has not been disclosed.

All cited references are incorporated herein by reference in theirentireties. The information disclosed herein is intended to assist theunderstanding of technical backgrounds of the present invention, andcannot be prior art.

DETAILED DESCRIPTION OF THE INVENTION

Technical Problem

The present invention provides a method of preparing piceatannol usingan enzyme which stably and efficiently catalyzes oxidation ofresveratrol into piceatannol.

Technical Solution

While searching for a method of preparing piceatannol, the presentinventors found that bacterial P450 (CYP102A1) and mutants thereof maybe selectively used to produce metabolites of resveratrol in humans,particularly piceatannol.

Advantageous Effects

The present invention provides a method of producing large quantities ofpiceatannol, which is about 60 times more expensive than resveratrol,from resveratrol, and a composition and a kit therefor.

While metabolites of resveratrol produced using the human CYP1A2 includepiceatannol and other hydroxylated products, piceatannol may beselectively produced by CYP102A1 or mutants thereof.

In addition, in an in vitro system, human CYP1A2 may be inactivated bythe metabolites of the human CYP1A2 itself. However, wild-type CYP102A1or mutants of CYP102A1 may not be inactivated by the metabolites.

Even though chemical synthesis of piceatannol has been reported,biological synthesis of piceatannol using enzymes is effective andenvironmentally friendly in terms of white biotechnology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates HPLC chromatograms of resveratrol metabolitesproduced by human CYP1A2 and bacterial CYP102A1 mutants (A: resveratroland piceatannol standards, B: human CYP1A2, C: Mutant #10, D: Mutant#13, E: Mutant #14, and F: Mutant #15). Peaks of the substrateresveratrol and two major products are indicated. UV absorbance wasmonitored at 320 nm.

FIG. 2 illustrates GC analysis results of resveratrol metabolitederivatives produced by CYP102A1 and mutants thereof (A:trans-resveratrol and piceatannol standards, B: human CYP1A2, C: Mutant#10, D: Mutant #13, E: Mutant #14, and F: Mutant #15). The mass spectraof the reaction samples showed peaks at 28.09 min (resveratrol) and32.98 min (piceatannol).

FIG. 3 illustrates GC elution profiles (A) and MS spectra (B:resveratrol, C and D: resveratrol metabolites) of resveratrol metabolitederivatives produced by human CYP1A2.

FIG. 4 illustrates MS spectra of peaks of metabolites produced bystandard trans-resveratrol (A) and piceatannol (B) that were eluted at29.08 min and 32.98 min (Res-TMS; m/z=444, Pic-TMS; m/z=532), peaks ofmetabolites produced by human CYP1A2 that were eluted at 29.08 min and32.98 min (C: Res-TMS; m/z=444, D: Pic-TMS; m/z=532), and peaks ofmetabolites produced by CYP102A1 mutants that were eluted at 32.98 min(Pic-TMS; m/z=532) (E: Mutant #10, F: Mutant #13, G: Mutant #14, and H:Mutant #15).

FIG. 5 illustrates total turnover numbers (TTN; mol product/molcatalyst) of piceatannol formation by CYP102A1 mutants. 100 μMtrans-resveratrol was used. The reaction was initiated by the additionof the NADPH-generating system, incubated for 1 or 2 hours,respectively, at 30° C. The formation rate of piceatannol was determinedby HPLC.

FIG. 6 illustrates the stability of P450 enzymes measured byCO-difference spectra during the oxidation of resveratrol by the P450enzymes in the presence of NADPH. Value of 100% represents the P450concentration before the incubation of the reaction mixture. Mutants#10, 11, 14, and 15 showed the highest stability.

FIG. 7 shows amino acid sequence of CYP102A1.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a method of preparing piceatannol, andmore particularly, a method of preparing piceatannol from resveratrolusing bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof, and acomposition and a kit therefor.

The present inventors have found that bacterial CYP102A1 and mutantsthereof may act as a catalyst for oxidation of resveratrol known as asubstrate of human P450. In particular, while metabolites of resveratrolproduced by the human CYP1A2, as a catalyst, include piceatannol andother hydroxylated products, piceatannol may be selectively produced byCYP102A1 or mutants thereof, as a catalyst. Although human CYP1A2 isinactivated in vitro by the metabolites, wild-type CYP102A1 or mutantsof CYP102A1 are not inactivated in vitro by the metabolites.

Specifically, the present inventors have identified thattrans-resveratrol is converted into hydroxylated metabolites when largequantities of wild-type CYP102A1 and site-directed mutants of CYP102A1are expressed in E. coli (Tables 1 and 2), and the wild-type and mutantsof CYP102A1 are subjected to a reaction with trans-resveratrol and anNADPH-generating system, using HPLC (FIG. 1) and GC-MS spectra (FIGS. 2to 5). The human CYP1A2 oxidizes resveratrol to produce two majormetabolites: piceatannol and other hydroxylated products. On the otherhand, wild-type CYP102A1 and mutants of CYP102A1 selectively produceonly one major metabolite: piceatannol.

Turnover numbers of trans-resveratrol oxidation (piceatannol formation)by CYP102A1 mutants are measured. As a result, mutants #8 to #17 showedhigher catalytic activities than that of wild-type CYP102A1. Mutant #13has about 18-fold higher activity than the wild-type CYP102A1 (Table 3).After 1 or 2 hours of hydroxylation of resveratrol by CYP102A1 mutants,the total turnover number (TTN; mol product/mol catalyst) of thepiceatannol formation is measured. As a result, mutant #13 shows thehighest activity and has about 10-fold higher activity than humanCYP1A1. The amount of products obtained by 1 hour-hydroxylation by humanCYP1A2 is less than that obtained by 2 hour-hydroxylation by humanCYP1A2. This is inferred because the human CYP1A2 is unstable or theactivity of the human CYP1A2 is inhibited by the metabolites.

Kinetic parameters for 3′-hydroxylation of resveratrol by wild-typeCYP102A1 and mutants of CYP102A1 are measured. Mutant #13 shows thehighest k_(cat) and K_(m) and the highest catalytic efficiency(k_(cat)/K_(m)) (Table 4). In case of human CYP1A2, kinetic parameterscould not be obtained. This is inferred because human P450 isinactivated by the metabolites of human P450 itself. Thus, human CYP1A2may not be used in an in vitro system, but wild-type CYP102A1 or mutantsof CYP102A1 may be used therein.

Resveratrol acts as a substrate and an inhibitor to human CYP1A2 at thesame time. Piceatannol, the major metabolite of resveratrol, is alsoknown as an inhibitor to human CYP1A2. Thus, the stability of P450enzymes is measured during oxidation of resveratrol by P450 enzymes inthe presence of NADPH using CO-difference spectra. As a result, mutants#10, 11, 14, and 15 show the highest stability (FIG. 6).

Based on these results, the present invention provides a composition forpreparing piceatannol from resveratrol, the composition includingwild-type CYP102A1 and/or mutants of CYP102A1.

The present invention also provides a method of preparing piceatannol,the method including reacting at least one enzyme selected from a groupconsisting of wild-type and mutants of CYP102A1 with resveratrol. Themethod may further include adding an NADPH-generating system.

The present invention also provides a kit for preparing piceatannol fromresveratrol, the kit including at least one enzyme selected from a groupconsisting of wild-type CYP102A1 and mutants of CYP10212 and anNADPH-generating system. The kit may further include a reagent requiredfor the progression of the reaction.

The NADPH-generating system used for the method and the kit may be anyknown system. For example, the NADPH-generating system may be glucose6-phosphate, NADP+, and yeast glucose 6-phosphate, but is not limitedthereto.

The piceatannol formation may be performed at a temperature ranging fromabout 0° C. to about 40° C., preferably from about 30° C. to about 40°C.

Mutagenesis of CYP102A1 may be performed using known methods such asdeletion mutagenesis (Kowalski D. et al., J. Biochem., 15, 4457), PCTmethod, Kunkel method, site-directed mutagenesis, DNA shuffling, StEP(staggered extension process), error-prone PCR, etc.

The mutants of CYP102A1 may have a sequence modified by natural orartificial substitution, deletion, addition, and/or insertion of aminoacid of the wild-type CYP102A1. The amino acid may be substituted withan amino acid with similar properties. For example, alanine, valine,leucine, isoleucine, proline, methionine, phenylalanine, and tryptophanare non-polar amino acids with similar properties. Neutral amino acidsare glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine, acidic amino acids are aspartic acid, glutamic acid, andbasic amino acids are lysine, arginine, and histidine.

The mutants of CYP102A1 include polypeptide with an amino acid sequencewhich is more than 50% similar, preferably more than 75% similar, andmore preferably more than 90% similar to the sequence of wild-typeCYP102A1.

The mutants of CYP102A1 may be prepared by at least one selected from agroup consisting of: substituting 47^(th) amino acid arginine (R) ofwild-type CYP102A1 with one amino acid selected from a group consistingof alanine, valine, leucine, isoleucine, proline, methionine,phenylalanine, and tryptophan, substituting 51^(st) amino acidtyrosine(Y) of wild-type CYP102A1 with one amino acid selected from agroup consisting of phenylalanine, alanine, valine, leucine, isoleucine,proline, methionine, tryptophan, substituting 64^(th) amino acidglutamic acid (E) of wild-type CYP102A1 with one amino acid selectedfrom a group consisting of glycine, serine, threonine, cysteine,tyrosine, asparagine, and glutamine, substituting 74^(th) amino acidalanine (A) of wild-type CYP102A1 with one amino acid selected from agroup consisting of glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine, substituting 81^(st) amino acid phenylalanine(F) of wild-type CYP102A1 with one amino acid selected from a groupconsisting of alanine, valine, leucine, isoleucine, proline, methionine,and tryptophan, substituting 86^(th) amino acid leucine (L) of wild-typeCYP102A1 with one amino acid selected from a group consisting ofalanine, valine, isoleucine, proline, methionine, phenylalanine, andtryptophan, substituting 87^(th) amino acid phenylalanine (F) ofwild-type CYP102A1 with one amino acid selected from a group consistingof alanine, valine, leucine, isoleucine, proline, methionine, andtryptophan, substituting 143^(rd) amino acid glutamic acid (E) ofwild-type CYP102A1 with one amino acid selected from a group consistingof glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine, substituting 188^(th) amino acid leucine (L) of wild-typeCYP102A1 with one amino acid selected from a group consisting ofglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine, and substituting 267^(th) amino acid glutamic acid (E) ofwild-type CYP102A1 with one amino acid selected from a group consistingof alanine, valine, leucine, isoleucine, proline, methionine,phenylalanine, and tryptophan.

Preferably, the mutants of CYP102A1 may be prepared by at least oneselected from a group consisting of: substituting 47^(th) amino acidarginine (R) of wild-type CYP102A1 with leucine (L), substituting51^(st) amino acid tyrosine(Y) of wild-type CYP102A1 with phenylalanine,substituting 64^(th) amino acid glutamic acid (E) of wild-type CYP102A1with glycine (G), substituting 74^(th) amino acid alanine (A) ofwild-type CYP102A1 with glycine (G), substituting 81^(st) amino acidphenylalanine (F) of wild-type CYP102A1 with isoleucine (I),substituting 86^(th) amino acid leucine (L) of wild-type CYP102A1 withisoleucine (I), substituting 87^(th) amino acid phenylalanine (F) ofwild-type CYP102A1 with valine (V), substituting 143^(rd) amino acidglutamic acid (E) of wild-type CYP102A1 with glycine (G), substituting188^(th) amino acid leucine (L) of wild-type CYP102A1 with glutamine(Q), and substituting 267^(th) amino acid glutamic acid (E) of wild-typeCYP102A1 with valine (V).

More preferably, the mutants of CYP102A1 may include amino acidsubstitution sites of wild-type CYP102A1 selected from a groupconsisting of F87A, R47L/Y51F, A74G/F87V/L188Q, R47L/L86I/L188Q,R47L/F87V/L188Q, R47L/F87V/L188Q/E267V, R47L/L86I/L188Q/E267V,R47L/L86I/F87V/L188Q, R47L/F87V/E143G/L188Q/E267V,R47L/E64G/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/L188Q/E267V, andR47L/E64G/F81I/F87V/E143G/L188Q/E267V.

Protein according to the present invention may be prepared using methodsknown in the art, for example, genetic engineering techniques,solid-phase peptide synthesis (Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)), or method of cleaving protein using peptidases.Protein according to the present invention may be natural protein, ormay be prepared by a recombination of culturing cells transformed withDNA encoding CYP102A1 or mutants thereof and collecting the protein.Protein may be prepared by inserting nucleic acid molecules encodingprotein according to the present invention into an expression vector,transforming the vector into a host cell, and purifying proteinexpressed by the transformed host cell.

The vector may be plasmid, cosmid, a virus, or phage. The host cell intowhich DNA in the vector is cloned or expressed may be a prokaryoticcell, a yeast cell, and a eukaryotic cell. Culture conditions such as aculture medium, temperature, and pH may be selected by those of ordinaryskill in the art without undue experiment. In general, principles,protocols, and techniques to maximize productivity of the culture ofcells are disclosed in Mammalian Cell Biotechnology: a PracticalApproach, M. Butler, ed. (IRL Press, 1991).

The expression and cloning vector may include a promoter operationallylinked to a nucleic acid sequence that encodes CYP102A2 or mutantsthereof which induce the synthesis of mRNA. Various promoters recognizedby host cells are known. A promoter suitable for a prokaryotic host cellmay be β-lactamase and a lactose promoter system, alkali phosphatase, atryptophan (trp) promoter system, and a hybrid promoter, for example atac promoter. In addition, the promoter used in bacterial systems mayinclude a Shine-Dalgarno (SD) sequence operationally linked to DNA thatencodes protein. A promoter suitable for a yeast host cell may include3-phosphoglycerate kinase or other glucosidases.

The present invention will now be described in greater detail withreference to the following examples, which are for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLES

Trans-resveratrol, trans-piceatannol, and NADPH were purchased fromSigma-Aldrich (Milwaukee, Wis., USA). Other chemicals were of thehighest grade commercially available. Human CYP1A2 was prepared asdisclosed by Kim et al., (2008c).

Example 1 Construction of P450 BM3 Mutants By Site-Directed Mutagenesis

Site-directed mutants of CYP102A1 were prepared as disclosed by Kim etal., Drug Metab Dispos, volume 35, p. 2166-2170, 2008. PCR primers usedto introduce BamHI/SacI restriction sites and to induce mutation arelisted in Table 1. Codons for amino acid substitution are in italics andunderlined. The PCR primers were obtained from Genotech (Daejeon,Korea). Genes that encode CYP102A1 mutants were amplified from pCWBM3 byPCR using primers designed to facilitate cloning into an expressionvector pCWori (Dr. F. W. Dahlquist, University of California, SantaBarbara, Calif.) or pSE420 (Invitrogen) (Farinas et al., 2001).Oligonucleotide assembly was performed by PCR using the 14 sets ofdesigned primers listed in Table 1. The amplified genes weresubsequently cloned into the PCWBM3 BamHI/SacI vector at the BamHI/SacIrestriction sites. These plasmids were transformed into Escherichia coliDH5a F′IQ (Invitrogen), and this strain was also used to express themutant CYP102A1 proteins. After mutagenesis, the presence of the desiredmutations was confirmed by DNA sequencing in Genotech (Daejeon, Korea).

TABLE 1 Primers used for the generation of mutants in this study NameSequence BamHI forward 5′-AGC  GGA TC C ATG ACA ATT AAA GAA ATG CCT C-3′SacI reverse 5′-ATC GAG CTC GTA GTT TGT AT-3′ R47L 5′-GCG CCT GGT  CTG GTA ACG CG-3′ Y51F 5′-GTA ACG CGC  TTC  TTA TCA AGT-3′ E64G5′-GCA TGC GAT  GGC  TCA CGC TTT-3′ A74G 5′-TA AGT CAA  GGC CTT AAA TTT GTA CG-3′ F81I 5′-GTA CGT GAT  ATT  GCA GGA GAC-3′ L86I5′-GGA GAC GGG  ATT  TTT ACA AGC T-3′ F87A 5′-GAC GGG TTA  GCG ACA AGC TGG-3′ F87V 5′-GAC GGG TTA  GTG  ACA AGC TGG-3′ E143G5′-GAA GTA CCG  GGC  GAC ATG ACA-3′ L188Q 5′-ATG AAC AAG  CAG CAG CGA GCA A-3′ A264G 5′-TTC TTA ATT  GGG  GGA CAC GTG-3′ E267V5′-T GCG GGA CAC  GTG  ACA ACA AGT-3′ L86I/F87V 5′-GGA GAC GGG  ATT GTG ACA AGC TG-3′

Example 2 Expression And Purification of Wild-Type CYP102A1 And Mutantsof CYP102A1

Plasmids comprising a gene of wild-type CYP102A1 and mutants of CYP102A1(pCWBM3) were transformed into Escherichia coli DH5α F′-IQ. A singlecolony was inoculated into 5 ml of a Luria-Bertani medium supplementedwith ampicillin (100 g/ml) and cultured at 37° C. This culture wasinoculated into 250 ml of a Terrific Broth medium supplemented withampicillin (100 g/ml). The cells were grown at 37° C. while shaking at250 rpm to an 0D₆₀₀ of up to 0.8, at which gene expression was inducedby the addition of isopropyl-β-D-thiogalactopyranoside to a finalconcentration of 0.5 mM. δ-Aminolevulinic acid (1.0 mM) was also addedthereto. Following induction, the cultures were allowed to grow another36 hours at 30° C. Cells were harvested by centrifugation (15 min, 5000g, 4° C.). The cell pellet was resuspended in a TES buffer (100 mMTris-HCl, pH 7.6, 500 mM sucrose, 0.5 mM EDTA) and lysed by sonication(Sonicator; Misonix, Inc., Farmingdale, N.Y.). After the lysate wascentrifuged at 100,000 g (90 min, 4° C.), a soluble cytosolic fractionwas collected and used for the activity assay. The soluble cytosolicfraction was dialyzed against a 50 mM potassium phosphate buffer (pH7.4) and stored at −80° C. Enzymes were used within 1 month ofmanufacture.

CYP102A1 concentrations were determined from the CO-difference spectraas described by Omura and Sato (1964) using ε=91 mM/cm. For all of thewild-type enzymes and mutated enzymes, a typical culture yielding of 300to 700 nM P450 enzymes could be detected. The expression level ofwild-type CYP102A1 and mutants of CYP102A1 was in the range of 1.0 to2.0 nmol P450/mg cytosolic protein.

Several mutants with high catalytic activity for humans were selected,and the substitution sites in the mutants are shown in Table 2 below.

TABLE 2 CYP102A1 mutants used in this study Abbreviations BM3 wild typeand mutants Ref. WT BM3 wild type Mutant #1 F87A Carmichael et al., 2001Mutant #2 A264G Carmichael et al., 2001 Mutant #3 F87A/A264G Carmichaelet al., 2001 Mutant #4 R47L/Y51F Carmichael et al., 2001 Mutant #5R47L/Y51F/A264G Carmichael et al., 2001 Mutant #6 R47L/Y51F/F87ACarmichael et al., 2001 Mutant #7 R47L/Y51F/F87A/A264G Carmichael etal., 2001 Mutant #8 A74G/F87V/L188Q Li et al., 2001 Mutant #9R47L/L86I/L188Q Kim et al., 2008a Mutant #10 R47L/F87V/L188Q vanVugt-Lussenburg et al., 2007 Mutant #11 R47L/F87V/L188Q/E267V vanVugt-Lussenburg et al., 2007 Mutant #12 R47L/L86I/L188Q/E267V Kim etal., 2008 Mutant #13 R47L/L86I/F87V/L188Q van Vugt-Lussenburg et al.,2007 Mutant #14 R47L/F87V/E143G/L188Q/E267V Kim et al., 2008a Mutant #15R47L/E64G/F87V/E143G/L188Q/E267V Kim et al., 2008a Mutant #16R47L/F81I/F87V/E143G/L188Q/E267V Kim et al., 2008a Mutant #17R47L/E64G/F81I/F87V/E143G/L188Q/E267V van Vugt-Lussenburg et al., 2007

Example 3 Hydroxylation of Trans-Resveratrol By Wild-Type P450 BM3 AndMutants of P450 BM3

Oxidation of trans-resveratrol, a substrate of human CYP1A2, by CYP102A1was identified. Typical steady-state reactions for trans-resveratrolhydroxylation included 50 pmol P450 BM3 in 0.25 ml of a 100 mM potassiumphosphate buffer (pH 7.4) were performed along with a specified amountof a substrate. To determine the kinetic parameter of several CYP102A1mutants, 2 to 100 μM of trans-resveratrol was used. An NADPH-generatingsystem was used to initiate reaction solutions (final concentrations: 10mM glucose 6-phosphate, 0.5 mM NADP+, and 1 IU yeast glucose 6-phosphateper ml). Trans-resveratrol stocks (20 mM) were prepared in DMSO anddiluted into the enzyme reactions with the final organic solventconcentration <1% (v/v). Reactions were generally incubated for 10 minat 37° C., and terminated with 105 μl of ice-cold acetic acid/methanol(95/5, v/v).

Example 3-1 HPLC Analysis

After centrifugation of the reaction mixture, the supernatant wasanalyzed by HPLC (Piver et al. 2004). Samples (30 μl) were injected intoa Gemini C₁₈ column (4.6 mm×150mm, 5 μm, Phenomenex, Torrance, Calif.).The mobile phase A was water containing 87 mM of 0.5% aceticacid/acetonitrile (95/5, v/v); whereas the mobile phase B wasacetonitrile/0.5% acetic acid (95/5, v/v). The mobile phase NB (75/25,v/v) was delivered at a flow rate of 1 ml·min⁻¹ by a gradient pump(LC-20AD, Shimadzu, Kyoto, Japan). Eluates were detected by UV rays at320 nm.

FIG. 1 illustrates HPLC chromatograms of resveratrol metabolitesproduced by human CYP1A2 and bacterial CYP102A1 mutants (A: resveratroland piceatannol standards, B: human CYP1A2, C: Mutant #10, D: Mutant#13, E: Mutant #14, and F: Mutant #15). Peaks of the substrateresveratrol and two major products are indicated. UV absorbance wasmonitored at 320 nm.

While human CYP1A2 oxidized resveratrol to produce two majormetabolites: piceatannol and other hydroxylated products (B), wild-typeCYP102A1 and mutants of CYP102A1 produced only one major metabolite. Theretention time of the peak was exactly matched to that of thepiceatannol standard. That is, the wild-type CYP102A1 and mutants ofCYP102A1 selectively produced piceatannol when oxidizing resveratrol.Since there is no need to separate the hydroxylated product frompiceatannol, the use of wild-type CYP102A1 and mutants of CYP102A1 isbeneficial.

Example 3-2 GC-MS Analysis

For the identification of resveratrol metabolite, produced by P450 BM3mutants, GC-MS analysis was done by comparing GC-profiles andfragmentation patterns of piceatannol and resveratrol. An oxidationreaction of trans-resveratrol by P450 BM3 mutants was done. The aqueoussamples were extracted with ethyl acetate. After centrifugation, theorganic phase was dried under nitrogen as well as the standardtrans-resveratrol and piceatannol solutions (10 mM in DMSO). Then,trimethylsilyl (TMS) derivatives were prepared as follows. 100 μl of asolution of BSTFA/TMCS (99/1, v/v) (Supelco) was added to the dryresidue or standard trans-resveratrol and piceatannol, and then themixture was left for 60 min at 60° C.

GC-MS analysis was performed on a GC-2010 gas chromatograph (Shimadzu,Kyoto, Japan) with an Rtx-5 (5% diphenyl/95% dimethyl polysiloxanecapillary column) (30 m×0.32 mm i.d.×0.25 pm film thickness). Theinjector temperature was 250° C. The derivatives of resveratrol andpiceatannol were separated by GC analysis under the conditions: GC ovenconditions of 60° C. for 5 min, followed by an increase of 50° C.·min⁻¹up to 200° C. and then 2° C.·min⁻¹ up to 300° C. The gas chromatographywas combined with a GCMS-QP2010 Shimazu mass spectrometer operating inan electron ionization mode (70 eV) (Piver et al. 2004).

FIG. 2 illustrates GC analysis results of resveratrol metabolitederivatives produced by CYP102A1 and mutants thereof (A: standardtrans-resveratrol and piceatannol, B: human CYP1A2, C: Mutant #10, D:Mutant #13, E: Mutant #14, and F: Mutant #15). The mass spectra of thereaction samples showed peaks at 28.09 min (resveratrol) and 32.98 min(piceatannol).

As a result of the GC-MS analysis, it was identified that the retentiontime and fragmentation patterns of the metabolite produced by theCYP102A1 mutants were exactly matched to those of the piceatannolstandard, and thus the metabolite produced by the CYP102A1 mutants waspiceatannol. Although piceatannol and other hydroxylated products werefound as the two major metabolites of resveratrol by human livermicrosomes, all wild-type CYP102A1 and mutants of CYP102A1 showed onlyone hydroxylated product, i.e., piceatannol. The human P450 1A2, themajor enzyme for hydroxylation reactions of resveratrol in human liver,also showed a preference for the 3′-hydroxylation reaction over thehydroxylation reaction at other position humans (B of FIG. 2 and FIG.4). However, unlike the human P450 enzyme, wild-type CYP102A1 andmutants of CYP102A1 produced only piceatannol without producing otherhydroxylated products.

FIG. 3 illustrates GC elution profiles (A) and MS spectra (B:resveratrol, C and D: resveratrol metabolites) of trans-resveratrolmetabolite derivatives produced by human CYP1A2.

FIG. 4 illustrates MS spectra of peaks of metabolites produced bystandard trans-resveratrol (A) and piceatannol (B) that were eluted at29.08 min and 32.98 min (Res-TMS; m/z=444, Pic-TMS; m/z=532), peaks ofmetabolites produced by human CYP1A2 that were eluted at 29.08 min and32.98 min (C: Res-TMS; m/z=444, D: Pic-TMS; m/z=532), and peaks ofmetabolites produced by CYP102A1 mutants that were eluted at 32.98 min(Pic-TMS; m/z=532) (E: Mutant #10, F: Mutant #13, G: Mutant #14, and H:Mutant #15).

Example 3-3 Determination of Total Turnover Number

To determine the total turnover number of CYP102A1 mutants, 100 μM oftrans-resveratrol was used. The reaction was initiated by the additionof the NADPH-generating system, incubated for 1 and 2 hours,respectively, at 30° C. The formation rate of piceatannol was determinedby HPLC as described above.

Table 3 shows turnover numbers of 17 mutants for trans-resveratroloxidation (piceatannol formation). The ability of wild-type P450 BM3 andmutants of P450 BM3 to oxidize trans-resveratrol was measured at a fixedsubstance concentration (100 μM).

TABLE 3 Rates of piceatannol formation by various CYP102A1 mutants^(a)nmol product/min/nmol P450 Enzyme Piceatannol WT 0.22 ± 0.01 Mutant #10.021 ± 0.001 #2 ND ^(b) #3 ND ^(b) #4 0.027 ± 0.001 #5 ND ^(b) #6 ND^(b) #7 ND ^(b) #8 1.1 ± 0.1 #9 0.24 ± 0.01 #10 1.6 ± 0.1 #11 0.64 ±0.03 #12 0.33 ± 0.03 #13 4.0 ± 0.1 #14 1.3 ± 0.1 #15 1.9 ± 0.3 #16 0.97± 0.1  #17 1.8 ± 0.1

Mutants #8 to #17 showed higher catalytic activities than that ofwild-type CYP102A1. Mutant #13 showed about 18-fold higher activity thanthe wild-type CYP102A1.

FIG. 5 illustrates total turnover numbers (TTN; mol product/molcatalyst) piceatannol formation by CYP102A1 mutants. 100 μMtrans-resveratrol was used. The reaction was initiated by the additionof the NADPH-generating system, incubated for 1 or 2 hours,respectively, at 30° C. The formation rate of piceatannol was determinedby HPLC as described above.

Mutant #13 showed the highest activity and has about 10-fold higheractivity than human CYP1A1. Meanwhile, the amount of products obtainedby 1 hour-hydroxylation by human CYP1A2 is less than that obtained by 2hour-hydroxylation by human CYP1A2. This was inferred because CYP1A2 isunstable or the activity of the human CYP1A2 is inhibited by themetabolites.

Example 3-4 Determination of Kinetic Parameters

Kinetic parameters (K_(m) and k_(cat)) were determined using nonlinearregression analysis with GraphPad PRISM software (GraphPad, San Diego,Calif., USA). The data were analyzed using the standard Michaelis-Mentenequation: v =k_(cat)[E][S]/([S]+K_(m)), where the velocity of thereaction is a function of the rate-limiting step in turnover (k_(cat)),the enzyme concentration ([E]), substrate concentration ([S]), and theMichaelis constant (K_(m)).

Table 4 shows kinetic parameters for 3′-hydroxylation of resveratrol bywild-type CYP102A1 and mutants of CYP102A1.

TABLE 4 Kinetic parameters of piceatannol formation by CYP102A1 mutantsPiceatannol formation P450 BM3 k_(cat) (min⁻¹) K_(m) (μM) k_(cat)/K_(m)Mutant #9 0.20 ± 0.02 66 ± 14 0.0030 ± 0.0007 Mutant #10 1.1 ± 0.1 30 ±13 0.037 ± 0.016 Mutant #11 0.12 ± 0.01 2.7 ± 0.7 0.044 ± 0.012 Mutant#12 0.13 ± 0.01 54 ± 11 0.0024 ± 0.0005 Mutant #13 6.7 ± 0.3 15 ± 3 0.45 ± 0.09 Mutant #14 0.58 ± 0.04 13 ± 3  0.046 ± 0.011 Mutant #15 0.54± 0.02 16 ± 2  0.038 ± 0.005

Mutant #13 showed the highest k_(cat) and K_(m) and the highestcatalytic efficiency (k_(cat)/K_(m)).

In case of human CYP1A2, kinetic parameters could not be obtained. Thisis inferred because P450 is inactivated by the metabolites of P450itself (Chun et al., 1999, 2001). Thus, human CYP1A2 may not be used inan in vitro system, but wild-type CYP102A1 or mutants of CYP102A1 may beused therein.

Example 4 Stability of CYP102A1 Mutants

It is known that metabolites of resveratrol are potent inhibitorsagainst human CYPs, i.e., CYP1A1, 1A2, and 1B1 (Chun et al., 1999,2001). Resveratrol acts as a substrate and inhibitor to human CYP1A2 atthe same time (Fairman et al., 2007; Piver et al., 2004). Piceatannol,the major metabolite of resveratrol, is also known as a potent inhibitorto human CYP1A2 (Mikstacka et al., 2006).

FIG. 6 illustrates the stability of P450 enzymes measured byCO-difference spectra during the oxidation of resveratrol by the P450enzymes in the presence of NADPH. Value of 100% represents the P450concentration before the incubation of the reaction mixture. Mutants#10, 11, 14, and 15 showed the highest stability.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

REFERENCES

Athar M, Back J H, Tang X, Kim K H, Kopelovich L, Bickers D R, Kim A L(2007) Resveratrol: a review of preclinical studies for human cancerprevention. Toxicol Appl Pharmaco. 224:274-283.

Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. JBiotechnol 124:128-145.

Carmichael A B and Wong L L (2001) Protein engineering of Bacillusmegaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons.Eur J Biochem 268:3117-3125.

Chun Y J, Kim S, Kim D, Lee S K, Guengerich F P. (2001) A new selectiveand potent inhibitor of human cytochrome P450 1B1 and its application toantimutagenesis. Cancer Res 61:8164-8170.

Chun Y J, Kim M Y, Guengerich F P. (1999) Resveratrol is a selectivehuman cytochrome P450 1A1 inhibitor. Biochem Biophys Res Commun262:20-24.

Di Nardo G, Fantuzzi A, Sideri A, Panicco P, Sassone C, Giunta C andGilardi G (2007) Wild-type CYP102A1 as a biocatalyst: turnover of drugsusually metabolised by human liver enzymes. J Biol Inorg Chem12:313-323.

Farinas E T, Schwaneberg U, Glieder A and Arnold F H (2001) Directedevolution of a cytochrome P450 monooxygenase for alkane oxidation.Advanced Synthesis & Catalysis 343:601-606.

Fairman D A, Collins C, Chapple S (2007) Progress curve analysis ofCYP1A2 inhibition: a more informative approach to the assessment ofmechanism-based inactivation Drug Metab Dispos 35:2159-2165.

Guengerich F P (2002) Cytochrome P450 enzymes in the generation ofcommercial products. Nat Rev Drug Discov 1:359-366.

Guengerich F P, Gillam E M, Shimada T (1996) New applications ofbacterial systems to problems in toxicology. Crit Rev Toxicol 26:551-583.

Johnson M D, Zuo H, Lee K, Trebley J P, Rae J M, Weatherman R V,Zeruesanay D, Flockhart D A, Skaar T C (2004) Pharmacologicalcharacterization of 4-hydroxy-N-desmethyl tamoxifen, a novel metaboliteof tamoxifen. Breast Cancer Res Treat 207:1-9.

Kim D H, Kim K H, Kim D H, Liu K H, Jung H C, Pan J G, Yun C H (2008a)Generation of human metabolites of 7-ethoxycoumarin by bacterialcytochrome P450 BM3. Drug Metab Dispos 36:2166-2170.

Kim Y H, Kwon H S, Kim D H, Cho H J, Lee H S, Jun J G, Park J H, Kim J K(2008b) Piceatannol, a stilbene present in grapes, attenuates dextransulfate sodium-induced colitis. Int Immunopharmacol 8:1695-1702.

Kim D H, Kim K H, Isin E M, Guengerich F P, Chae H Z, Ahn T, Yun C H.(2008c) Heterologous expression and characterization of wild-type humancytochrome P450 1A2 without conventional N-terminal modification inEscherichia coli. Protein Expr Purif. 57:188-200.

Kundu J K, Surh Y J (2008) Cancer chemopreventive and therapeuticpotential of resveratrol: mechanistic perspectives. Cancer Lett269:243-261.

Lamb D C, Waterman M R, Kelly S L and Guengerich F P (2007) CytochromesP450 and drug discovery. Curr Opin Biotechnol 18:504-512.

Li Q S, Ogawa J, Schmid R D and Shimizu S (2001) Engineering cytochromeP450 BM-3 for oxidation of polycyclic aromatic hydrocarbons. ApplEnviron Microbiol 67:5735-5739.

Mikstacka R, Rimando A M, Szalaty K, Stasik K, Baer-Dubowska W (2006)Effect of natural analogues of trans-resveratrol on cytochromes P4501A2and 2E1 catalytic activities. Xenobiotica 36:269-285.

Narhi L O and Fulco A J (1982) Phenobarbital induction of a solublecytochrome P-450-dependent fatty acid monooxygenase in Bacillusmegaterium. J. Biol. Chem. 257:2147-150.

Omura T and Sato R (1964) The carbon monoxide-binding pigment of livermicrosomes. II. Solubilization, purification, and properties. J BiolChem 239:2379-2385.

Otey C R, Bandara G, Lalonde J, Takahashi K and Arnold F H (2005)Preparation of human metabolites of propranolol using laboratory-evolvedbacterial cytochrome P450. Biotechnol Bioeng 93:494-499.

Parikh A, Gillam E M, Guengerich F P (1997) Drug metabolism byEscherichia coli expressing human cytochromes P450. Nat Biotechnol15:784-788.

Pirola L, Frojdo (2008) Resveratrol: one molecule, many targets. IUBMBLife 60:323-332.

Potter G A, Patterson L H, Wanogho E, Perry P J, Butler P C, Ijaz T,Ruparelia K C, Lamb J H, Farmer P B, Stanley L A, Burke M D (2002) Thecancer preventative agent resveratrol is converted to the anticanceragent piceatannol by the cytochrome P450 enzyme CYP1B1. Br J Cancer86:774-778.

Piver B, Fer M, Vitrac X, Merillon J M, Dreano Y, Berthou F, Lucas D.(2004) Involvement of cytochrome P450 1A2 in the biotransformation oftrans-resveratrol in human liver microsomes. Biochem Pharmacol68:773-782.

Rushmore T H, Reider P J, Slaughter D, Assang C, Shou M (2000)Bioreactor systems in drug metabolism: Synthesis of cytochromeP450-generated metabolites. Metab Eng 2:115-125.

Stottmeister U, Aurich A, Wilde H, Andersch J, Schmidt S, Sicker D(2005) White biotechnology for green chemistry: fermentative2-oxocarboxylic acids as novel building blocks for subsequent chemicalsyntheses. J Ind Microbiol Biotechnol 32:651-664.

Urlacher V B and Eiben S (2006) Cytochrome P450 monooxygenases:perspectives for synthetic application. Trends Biotechnol 24:324-330.

Vail R B, Homann M J, Hanna I, Zaks A (2005) Preparative synthesis ofdrug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 withNADPH-P450 reductase expressed in Escherichia coli. J Ind MicrobiolBiotechnol 32:67-74.

van Vugt-Lussenburg B M, Damsten M C, Maasdijk D M, Vermeulen N P andCommandeur J N (2006) Heterotropic and homotropic cooperativity by adrug-metabolizing mutant of cytochrome P450 BM3. Biochem Biophys ResCommun 346:810-818.

van Vugt-Lussenburg B M, Stjernschantz E, Lastdrager J, Oostenbrink C,Vermeulen N P and Commandeur J N (2007) Identification of criticalresidues in novel drug-metabolizing mutants of cytochrome P450 BM3 usingrandom mutagenesis. J Med Chem 50:455-461.

Yun C H, Kim K H, Kim D H, Jung H C and Pan J G (2007) The bacterialP450 BM3: a prototype for a biocatalyst with human P450 activities.Trends Biotechnol 25:289-298.

1. A composition for a catalyst in the reaction of preparing piceatannolfrom resveratrol, the composition comprising at least one selected froma group consisting of wild-type CYP102A1 and mutants of CYP102A1.
 2. Thecomposition of claim 1, wherein the mutants of CYP102A1 are prepared byat least one selected from a group consisting of: substituting 47^(th)amino acid arginine (R) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, leucine,isoleucine, proline, methionine, phenylalanine, and tryptophan,substituting 51^(st) amino acid tyrosine(Y) of wild-type CYP102A1 withone amino acid selected from a group consisting of phenylalanine,alanine, valine, leucine, isoleucine, proline, methionine, tryptophan,substituting 64^(th) amino acid glutamic acid (E) of wild-type CYP102A1with one amino acid selected from a group consisting of glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine, substituting74^(th) amino acid alanine (A) of wild-type CYP102A1 with one amino acidselected from a group consisting of glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine, substituting 81^(st)amino acid phenylalanine (F) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, leucine,isoleucine, proline, methionine, and tryptophan, substituting 86^(th)amino acid leucine (L) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, isoleucine,proline, methionine, phenylalanine, and tryptophan, substituting 87^(th)amino acid phenylalanine (F) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, leucine,isoleucine, proline, methionine, and tryptophan, substituting 143^(rd)amino acid glutamic acid (E) of wild-type CYP102A1 with one amino acidselected from a group consisting of glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine, substituting 188^(th)amino acid leucine (L) of wild-type CYP102A1 with one amino acidselected from a group consisting of glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine, and substituting 267^(th)amino acid glutamic acid (E) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, leucine,isoleucine, proline, methionine, phenylalanine, and tryptophan.
 3. Thecomposition of claim 2, wherein the mutants of CYP102A1 are prepared byat least one selected from a group consisting of: substituting 47^(th)amino acid arginine (R) of wild-type CYP102A1 with leucine (L),substituting 51^(st) amino acid tyrosine(Y) of wild-type CYP102A1 withphenylalanine, substituting 64^(th) amino acid glutamic acid (E) ofwild-type CYP102A1 with glycine (G), substituting 74^(th) amino acidalanine (A) of wild-type CYP102A1 with glycine (G), substituting 81^(st)amino acid phenylalanine (F) of wild-type CYP102A1 with isoleucine (I),substituting 86^(th) amino acid leucine (L) of wild-type CYP102A1 withisoleucine (I), substituting 87^(th) amino acid phenylalanine (F) ofwild-type CYP102A1 with valine (V), substituting 143^(rd) amino acidglutamic acid (E) of wild-type CYP102A1 with glycine (G), substituting188^(th) amino acid leucine (L) of wild-type CYP102A1 with glutamine(Q), and substituting 267^(th) amino acid glutamic acid (E) of wild-typeCYP102A1 with valine (V).
 4. The composition of claim 1, wherein themutants of CYP102A1 comprises amino acid substitution sites of wild-typeCYP102A1 selected from a group consisting of F87A, R47L/Y51 F,A74G/F87V/L188Q, R47L/L86I/L188Q, R47L/F87V/L188Q,R47L/F87V/L188Q/E267V, R47L/L86I/L188Q/E267V, R47L/L86I/F87V/L188Q,R47L/F87V/E143G/L188Q/E267V, R47L/E64G/F87V/E143G/L188Q/E267V,R47L/F81I/F87V/E143G/L188Q/E267V, andR47L/E64G/F81I/F87V/E143G/L188Q/E267V.
 5. The composition of claim 1,wherein the resveratrol is trans-resveratrol.
 6. A method of preparingpiceatannol, the method comprising reacting at least one enzyme selectedfrom a group consisting of wild-type CYP102A1 and mutants of CYP102A1with resveratrol.
 7. The method of claim 6, further comprising adding anNADPH-generating system.
 8. The method of claim 7, wherein theNADPH-generating system comprises glucose 6-phosphate, NADP+, and yeastglucose 6-phosphate.
 9. The method of claim 6, wherein the resveratrolis trans-resveratrol.
 10. The method of claim 6, wherein the mutants ofCYP102A1 are prepared by at least one selected from a group consistingof: substituting 47^(th) amino acid arginine (R) of wild-type CYP102A1with one amino acid selected from a group consisting of alanine, valine,leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan,substituting 51^(st) amino acid tyrosine(Y) of wild-type CYP102A1 withone amino acid selected from a group consisting of phenylalanine,alanine, valine, leucine, isoleucine, proline, methionine, tryptophan,substituting 64^(th) amino acid glutamic acid (E) of wild-type CYP102A1with one amino acid selected from a group consisting of glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine, substituting74^(th) amino acid alanine (A) of wild-type CYP102A1 with one amino acidselected from a group consisting of glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine, substituting 81^(st)amino acid phenylalanine (F) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, leucine,isoleucine, proline, methionine, and tryptophan, substituting 86^(th)amino acid leucine (L) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, isoleucine,proline, methionine, phenylalanine, and tryptophan, substituting 87^(th)amino acid phenylalanine (F) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, leucine,isoleucine, proline, methionine, and tryptophan, substituting 143^(rd)amino acid glutamic acid (E) of wild-type CYP102A1 with one amino acidselected from a group consisting of glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine, substituting 188^(th)amino acid leucine (L) of wild-type CYP102A1 with one amino acidselected from a group consisting of glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine, and substituting 267^(th)amino acid glutamic acid (E) of wild-type CYP102A1 with one amino acidselected from a group consisting of alanine, valine, leucine,isoleucine, proline, methionine, phenylalanine, and tryptophan.
 11. Themethod of claim 6, wherein the mutants of CYP102A1 are prepared by atleast one selected from a group consisting of: substituting 47^(th)amino acid arginine (R) of wild-type CYP102A1 with leucine (L),substituting 51^(st) amino acid tyrosine(Y) of wild-type CYP102A1 withphenylalanine, substituting 64^(th) amino acid glutamic acid (E) ofwild-type CYP102A1 with glycine (G), substituting 74^(th) amino acidalanine (A) of wild-type CYP102A1 with glycine (G), substituting 81^(st)amino acid phenylalanine (F) of wild-type CYP102A1 with isoleucine (I),substituting 86^(th) amino acid leucine (L) of wild-type CYP102A1 withisoleucine (I), substituting 87^(th) amino acid phenylalanine (F) ofwild-type CYP102A1 with valine (V), substituting 143^(rd) amino acidglutamic acid (E) of wild-type CYP102A1 with glycine (G), substituting188^(th) amino acid leucine (L) of wild-type CYP102A1 with glutamine(Q), and substituting 267^(th) amino acid glutamic acid (E) of wild-typeCYP102A1 with valine (V).
 12. The method of claim 6, wherein the mutantsof CYP102A1 comprises amino acid substitution sites of wild-typeCYP102A1 selected from a group consisting of F87A, R47L/Y51 F,A74G/F87V/L188Q, R47L/L86I/L188Q, R47L/F87V/L188Q,R47L/F87V/L188Q/E267V, R47L/L86I/L188Q/E267V, R47L/L86I/F87V/L188Q,R47L/F87V/E143G/L188Q/E267V, R47L/E64G/F87V/E143G/L188Q/E267V,R47L/F81I/F87V/E143G/L188Q/E267V, andR47L/E64G/F81I/F87V/E143G/L188Q/E267V.
 13. A kit for preparingpiceatannol from resveratrol, the kit comprising at least one enzymeselected from a group consisting of wild-type CYP102A1 and mutants ofCYP102A1, and an NADPH-generating system.
 14. The kit of claim 13,wherein the NADPH-generating system comprises glucose 6-phosphate,NADP+, and yeast glucose 6-phosphate.